Sequence requirement for hand-in-hand interaction in formation of RNA dimers and hexamers to gear phi29 DNA translocation motor.

Translocation of DNA or RNA is a ubiquitous phenomenon. One intricate translocation process is viral DNA packaging. During maturation, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into a limited space within the procapsid. We have revealed that phi29 DNA packaging is accomplished by a mechanism similar to driving a bolt with a hex nut, which consists of six DNA-packaging pRNAs. Four bases in each of the two pRNA loops are involved in RNA/RNA interactions to form a hexagonal complex that gears the DNA translocating machine. Without considering the tertiary interaction, in some cases only two G/C pairs between the interacting loops could provide certain pRNAs with activity. When all four bases were paired, at least one G/C pair was required for DNA packaging. The maximum number of base pairings between the two loops to allow pRNA to retain wild-type activity was five, whereas the minimum number was five for one loop and three for the other. The findings were supported by phylogenetic analysis of seven pRNAs from different phages. A 75-base RNA segment, bases 23-97, was able to form dimer, to interlock into the hexamer, to compete with full-length pRNA for procapsid binding, and therefore to inhibit phi29 assembly in vitro. Our result suggests that segment 23-97 is a self-folded, independent domain involved in procapsid binding and RNA/RNA interaction in dimer and hexamer formation, whereas bases 1-22 and 98-120 are involved in DNA translocation but dispensable for RNA/RNA interaction. Therefore, this 75-base RNA could be a model for structural studies in RNA dimerization.     

Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.     

Boundary of pRNA functional domains and minimum pRNA sequence requirement for specific connector binding and DNA packaging of phage phi29.

Bacteriophage phi29 utilizes a viral-encoded 120-base RNA (pRNA) to accomplish dsDNA packaging into a preformed procapsid. Six pRNAs bind to the procapsid and work sequentially. The pRNA contains two functional domains, one for binding to the DNA translocating connector, and the other for interacting with another component of the DNA packaging machinery during DNA translocation. By UV crosslinking, the pRNA was found to bind to the connector specifically and not to the capsid or scaffolding proteins. When purified connectors were incubated with pRNA, rosette-like connector oligomers were observed. These oligomers were found to contain pRNA. A series of deletion mutants of the pRNA were constructed and their ability to perform various tasks involved in phi29 assembly were assayed. The minimum sizes of the pRNA needed for the following activities have been determined: (1) specific binding to procapsid or to connectors; (2) connector or procapsid binding with full efficiency compared with wild-type pRNA; and (3) genomic DNA packaging. In summary, bases 37-91 (55 nt) comprised the minimum sequence required for specific connector binding, although with lower efficiency; bases 6-113 (105 nt with the additional deletion of two nonessential bases, C109 and A106) comprised the minimum sequence required for full connector binding activity; and bases 1-117 comprised the minimum sequence needed for full DNA packaging activity. These data indicate clearly that the helical region composed of bases 1-6 and 113-117 plays a crucial role in DNA translocation, but is dispensable for connector binding. A model for the role of the pRNA in DNA packaging was also presented.     

A dimer as a building block in assembling RNA. A hexamer that gears bacterial virus phi29 DNA-translocating machinery

Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryo-atomic force microscopy revealed that monomers displayed a check mark outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg(2+), Ca(2+), and Mn(2+). Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer --> tetramer --> hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.     

Three-dimensional interaction of Phi29 pRNA dimer probed by chemical modification interference, cryo-AFM, and cross-linking

Six pRNAs (p for packaging) of bacterial virus phi29 form a hexamer complex that is an essential component of the viral DNA translocating motor. Dimers, the building block of pRNA hexamer, assemble in the order of dimer --> tetramer --> hexamer. The two-dimensional structure of the pRNA monomer has been investigated extensively; however, the three-dimensional structure concerning the distance constraints of the three stems and loops are unknown. In this report, we probed the three-dimensional structure of pRNA monomer and dimer by photo affinity cross-linking with azidophenacyl. Bases 75-81 of the left stem were found to be oriented toward the head loop and proximate to bases 26-31 in a parallel orientation. Chemical modification interference indicates the involvement of bases 45-71 and 82-91 in dimer formation. Dimer was formed via hand-in-hand contact, a novel RNA dimerization that in some aspects is similar to the kissing loops of the human immunodeficiency virus. The covalently linked dimers were found to be biologically active. Both the native dimer and the covalently linked dimer were found by cryo-atomic force microscopy to be similar in global conformation and size.     

Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29.

A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging. This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops. Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging. The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated. Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101. A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints. Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly. Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities. However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly. Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery. Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other packaging components.     

Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation.

Bacteriophage phi29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during maturation. An intriguing feature of phi29 assembly is that a virus-encoded RNA (pRNA) is required for the packaging of its genomic DNA. Psoralen cross-linking, primer extension, and T1 RNase partial digestion revealed that pRNA had at least two conformations; one was able to bind procapsids, and the other was not. In the presence of Mg2+, one stretch of pRNA, consisting of bases 31 to 35, was confirmed to be proximal to base 69, as revealed by its efficient cross-linking by psoralen. Two cross-linking sites in the helical region were identified. Mg2+ induced a conformational change of pRNA that exposes the portal protein binding site by promoting the refolding of two strands of the procapsid binding region, resulting in the formation of pRNA-procapsid complexes. The procapsid binding region in this binding-competent conformation could not be cross-linked with psoralen. When the two strands of the procapsid binding region were fastened by cross-linking, pRNA could neither bind procapsids nor package phi29 DNA. A pRNA conformational change was also discernible by comparison of migration rates in native EDTA and Mg2+ polyacrylamide gel electrophoresis and was revealed by T1 RNase probing. The Mg2+ concentration required for the detection of a change in pRNA cross-linking patterns was 1 mM, which was the same as that required for pRNA-procapsid complex formation and DNA packaging and was also close to that in normal host cells.     

pRNA的结构、功能及其研究近况

在噬菌体phi29中, 基因组DNA的包装需要由病毒基因组编码的pRNA参与, 6个pRNA分子通过由pRNA分子间相互作用形成的六聚体来启动DNA转运马达, 这个过程由ATP提供能量。RNA纳米技术将pRNA与siRNA、核酶、反义RNA等分子稳定结合, pRNA作为一种载体把它们准确运输到癌细胞和病毒感染细胞的作用靶点, 从而发挥它们各自的功能。作为一种非编码RNA, 对pRNA的深入研究将有助于我们了解生命起源问题, 并有着广阔的应用前景。     

Confirmation of the helical structure of the 5'/3' termini of the essential DNA packaging pRNA of phage phi 29.

Bacteriophage phi 29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during viral assembly. An intriguing feature of phi 29 is the presence of a 120-base virus-encoded RNA (pRNA) that is indispensable for DNA packaging. Phylogenetic comparison of similar RNAs in numerous phages has revealed that the secondary structure of the pRNA is well conserved. Computer analysis predicts the presence of an extensive segment of helix with three single-base bulges generated by the pairing of the 5' and 3' ends. The desire to understand the role played by the pRNA in DNA packaging has led to a mutational analysis of the 5'-/3'-terminal region, which is believed to be important in DNA translocation. Deletion of 3 bases from the 3' end of the RNA, shortening the pRNA from 120 to 117 bases, was tolerated without loss of activity, but additional deletion of the base 117 resulted in 100-fold less activity, and a 115-base pRNA was virtually nonfunctional. Additionally, the three unpaired one-base bulges within the helical stretches of the paired proximate ends were nonessential for pRNA activity, as demonstrated by deletion of the bulge individually. An extensive series of helix disruptions by single- and multiple-base substitution almost invariably led to the loss of DNA packaging activity. Additional mutations that restored predicted base pairings rescued pRNA activity. This second site suppression confirmed that the 5'- and 3'-end region was paired and was indeed a helical stretch. The secondary structure was of greater importance than the primary sequence, with the exception of the requirement of an adenine at either the third or fourth position. The specific requirement of an adenine in phi 29 pRNA at this position, as well as conservation of this position in other phage pRNAs, implicates that this base may play a special role in either the DNA-packaging reaction or the maintenance of the pRNA tertiary structure.     

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg²⁺. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.     

Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29

During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864–3871] suggested that the foothold for pRNA was the connector and that the pRNA–connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS–PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA–connector complex and that the foothold for pRNA is the connector but not the capsid protein.     

Sequential action of six virus-encoded DNA-packaging RNAs during phage phi29 genomic DNA translocation.

A 120-base pRNA encoded by bacteriophage b29 has a novel and essential role in genomic DNA packaging. Six DNA-packaging RNAs (pRNAs) were bound to the sixfold symmetrical portal vertex of procapsids during the DNA translocation process and left the procapsid after the DNA-packaging reaction was completed, suggesting that the pRNA participated in the translocation of genomic DNA into procapsids. To further investigate the mechanism of DNA packaging, it is crucial to determine whether these six pRNA molecules work as an integrated entity or each pRNA acts as a functional individual. If pRNAs work individually, then do they work in sequence with communication or in random order without interaction? Results from compensation and complementation analysis did not support the integrated model. Computation of the probability of combination between wild-type and mutant pRNAs and experimental data of competitive inhibition excluded the random model while favoring the proposal that the six pRNAs functioned sequentially. Sequential action of the pRNA also explains why the pRNA is so sensitive to mutation, since the effect of a pRNA mutation will be amplified by 6 orders of magnitude after six consecutive steps, resulting in the observed complete loss of DNA-packaging activity caused by small alterations. When any one of the six pRNAs was replaced with an inactive one, complete blockage of DNA packaging resulted, strongly supporting the speculation that individual pRNAs, presumably together with other components such as the packaging ATPase gp16, take turns mediating successive steps of packaging. Although the data provided here could not exclude the integrated model completely, there is no evidence so far to argue against the model of sequential action.     

Approaches to determine stoichiometry of viral assembly components.

Due to the rapidity of biological reactions, it is difficult to isolate intermediates or to determine the stoichiometry of participants in intermediate reactions. Instead of determining the absolute amount of each component, this study involved the use of relative parameters, such as dilution factors, percentages probabilities, and slopes of titration curves, that can be more accurately quantified to determine the stoichiometry of components involved in bacteriophage phi29 assembly. This work takes advantage of the sensitive in vitro phage phi29 assembly system, in which 10(8) infectious virions per ml without background can be assembled from eight purified components. It provides a convenient assay for quantification of the stoichiometry of packaging components, including the viral procapsid, genomic DNA, DNA-packaging pRNA, and other structural proteins and enzymes. The presence of a procapsid binding domain and another essential functional domain within the pRNA makes it an ideal component for constructing lethal mutants for competitive procapsid binding. Two methods were used for stoichiometry determination. Method 1 was to determine the combination probability of mutant and wild-type pRNAs bound to procapsids. The probability of procapsids that possess a certain amount of mutant and a certain amount of wild-type pRNA, both with an equal binding affinity, was predicted with the binomial equation [EQUATION IN TEXT] where Z is the total number of pRNAs per procapsid, M is the number of mutant pRNAs bound to one procapsid, and (ZM) is equal to [FORMULA IN TEXT]. With various ratios of mutant to wild-type pRNA in in vitro viral assembly, the percent mutant pRNA versus the yield of virions was plotted and compared to a series of predicted curves to find a best fit. It was determined that five or six copies of pRNA were required for one DNA-packaging event, while only one mutant pRNA per procapsid was sufficient to block packaging. Method 2 involved the comparison of slopes of curves of dilution factors versus the yield of virions. Components with known stoichiometries served as standard controls. The larger the stoichiometry of the component, the more dramatic the influence of the dilution factor on the reaction. A slope of 1 indicates that one copy of the component is involved in the assembly of one virion. A slope larger than 1 would indicate multiple-copy involvement. By this method, the stoichiometry of gp11 in phi29 particles was determined to be approximately 12. These approaches are useful for the determination of the stoichiometry of functional units involved in viral assembly, be they single molecules or oligomers. However, these approaches are not suitable for the determination of exact copy numbers of individual molecules involved if the functional unit is composed of multiple subunits prior to assembly.     

Controlling bacteriophage phi29 DNA-packaging motor by addition or discharge of a peptide at N-terminus of connector protein that interacts with pRNA

Bacteriophage phi29 utilizes a motor to translocate genomic DNA into a preformed procapsid. The motor contains six pRNAs, an enzyme and one 12-subunit connector with a central channel for DNA transportation. A 20-residue peptide containing a His-tag was fused to the N-terminus of the connector protein gp10. This fusion neither interfered with procapsid assembly nor affected the morphology of the prolate-shaped procapsid. However, the pRNA binding and virion assembly activity were greatly reduced. Such decreased functions can be switched back on by the removal of the tag via protease cleavage, supporting the previous finding that the N-terminus of gp10 is essential for the pRNA binding. The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA. It is speculated that the fusion of the tag generated physical hindrance to pRNA binding, with greater influence for the dimers than the monomers due to their size. These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.     

细菌病毒phi29DNA—装运泵六聚体RNA结构和功能的研究方法

在双链DNA病毒增殖和成熟的过程中 ,需要将相当长的子代DNA装入一个极为有限空间的新生病毒衣壳。整个核酸装壳过程是耗能的过程 ,必需依靠生物泵来将DNA推入壳中。在细菌病毒phi2 9的核酸装壳过程中 ,需要RNA分子作为此生物泵的重要构成组分。6个RNA分子构成一个六边形样螺帽 ,将DNA如螺栓般装入病毒衣壳。6个RNA的这种依次运动的轮流作用模型如同汽车发动机的 6个气缸依次起火的原理一样 ,只是能源来自ATP而不是汽油。综述了此RNA的结构 ,及其结构对其功能所起的重要作用 ,并着重阐述研究 pRNA结构的独特构思和方法     

Diverse self-association properties within a family of phage packaging RNAs

The packaging RNA (pRNA) found in phi29 bacteriophage is an essential component of a molecular motor that packages the phage''s DNA genome. The pRNA forms higher-order multimers by intermolecular “kissing” interactions between identical molecules. The phi29 pRNA is a proven building block for nanotechnology and a model to explore the rare phenomenon of naturally occurring RNA self-association. Although the self-association properties of the phi29 pRNA have been extensively studied and this pRNA is used in nanotechnology, the characteristics of phylogenetically related pRNAs with divergent sequences are comparatively underexplored. These diverse pRNAs may lend new insight into both the rules governing RNA self-association and for RNA engineering. Therefore, we used a combination of biochemical and biophysical methods to resolve ambiguities in the proposed secondary structures of pRNAs from M2, GA1, SF5, and B103 phage, and to discover that different naturally occurring pRNAs form multimers of different stoichiometry and thermostability. Indeed, the M2 pRNA formed multimers that were particularly thermostable and may be more useful than phi29 pRNA for many applications. To determine if diverse pRNA behaviors are conferred by different kissing loop sequences, we designed and tested chimeric RNAs based on our revised secondary structural models. We found that although the kissing loops are essential for self-association, the critical determinant of multimer stability and stoichiometry is likely the diverse three-way junctions found in these RNAs. Using known features of RNA three-way junctions and solved structures of phi29 pRNA''s junction, we propose a model for how different junctions affect self-association.     

The procapsid binding domain of phi29 packaging RNA has a modular architecture and requires 2'-hydroxyl groups in packaging RNA interaction

The phi29 packaging RNA (pRNA) is an essential component in the phi29 bacteriophage DNA packaging motor, the strongest biomolecular motor known today. Utilizing Mg2+-dependent intermolecular base pairing interactions between two 4-nucleotide loops within the pRNA procapsid binding domain, multiple copies of pRNA form a ring-shaped complex that is indispensable for packaging motor function. To understand pRNA structural organization and pRNA/pRNA interaction, studies were carried out on pRNA closed dimers, the simplest functional pRNA complex believed to be the building blocks for assembling the oligomeric ring. Tertiary folding and interactions in various pRNA mutants were evaluated based on measured closed dimer affinity that is directly linked to the proper positioning of the interacting loops. The data revealed that the procapsid binding domain contains two autonomous modules that are capable of interacting noncovalently to form a fully active species in pRNA/pRNA interaction. Deleting the 2'-hydroxyl groups in one of the interacting loops weakens the dimer affinity by 125-fold, suggesting potential tertiary interactions involving these 2'-hydroxyl groups. The results provide evidence that nonbase functional groups are involved in pRNA folding and interaction and lead to a simple model that describes the pRNA monomer configuration in terms of three arms spanning a hinge. The functional constructs developed here will aid biophysical and biochemical investigations of pRNA structure and function, as well as developments of pRNA-based technology for nanoscience and gene therapy.     

Interaction of gp16 with pRNA and DNA for genome packaging by the motor of bacterial virus phi29

One striking feature in the assembly of linear double-stranded (ds) DNA viruses is that their genome is translocated into a preformed protein coat via a motor involving two non-structural components with certain characteristics of ATPase. In bacterial virus phi29, these two components include the protein gp16 and a packaging RNA (pRNA). The structure and function of other phi29 motor components have been well elucidated; however, studies on the role of gp16 have been seriously hampered by its hydrophobicity and self-aggregation. Such problems caused by insolubility also occur in the study of other viral DNA-packaging motors. Contradictory data have been published regarding the role and stoichiometry of gp16, which has been reported to bind every motor component, including pRNA, DNA, gp3, DNA-gp3, connector, pRNA-free procapsid, and procapsid/pRNA complex. Such conflicting data from a binding assay could be due to the self-aggregation of gp16. Our recent advance to produce soluble and highly active gp16 has enabled further studies on gp16. It was demonstrated in this report that gp16 bound to DNA non-specifically. gp16 bound to the pRNA-containing procapsid much more strongly than to the pRNA-free procapsid. The domain of pRNA for gp16 interaction was the 5'/3' paired helical region. The C18C19A20 bulge that is essential for DNA packaging was found to be dispensable for gp16 binding. This result confirms the published model that pRNA binds to the procapsid with its central domain and extends its 5'/3' DNA-packaging domain for gp16 binding. It suggests that gp16 serves as a linkage between pRNA and DNA, and as an essential DNA-contacting component during DNA translocation. The data also imply that, with the exception of the C18C19A20 bulge, the main role of the 5'/3' helical double-stranded region of pRNA is not for procapsid binding but for binding to gp16.     

Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging

Many nucleic acid-binding proteins and the AAA+ family form hexameric rings, but the mechanism of hexamer assembly is unclear. It is generally believed that the specificity in protein/RNA interaction relies on molecular contact through a surface charge or 3D structure matching via conformational capture or induced fit. The pRNA of bacteriophage phi29 DNA-packaging motor also forms a ring, but whether the pRNA ring is a hexamer or a pentamer is under debate. Here, single molecule studies elucidated a mechanism suggesting the specificity and affinity in protein/RNA interaction relies on pRNA static ring formation. A combined pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group bind to the motor nonspecifically. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension or reduction of the ring circumference failed in motor binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and packaged DNA. The results confirmed the stoichiometry of pRNA on the motor was the common multiple of two and three, thus, a hexamer.     

Complete inhibition of virion assembly in vivo with mutant procapsid RNA essential for phage phi 29 DNA packaging.

A highly efficient method for the inhibition of bacteriophage phi 29 assembly was developed with the use of mutant forms of the viral procapsid (or packaging) RNA (pRNA) indispensable for phi 29 DNA packaging. Phage phi 29 assembly was severely reduced in vitro in the presence of mutant pRNA and completely blocked in vivo when the host cell expressed mutant pRNA. Addition of 45% mutant pRNA resulted in a reduction of infectious virion production by 4 orders of magnitude, indicating that factors involved in viral assembly can be targets for efficient and specific antiviral treatment. The mechanism leading to the high efficiency of inhibition was attributed to two pivotal features. First, the pRNA contains two separate, essential functional domains, one for procapsid binding and the other for a DNA-packaging role other than procapsid binding. Mutation of the DNA-packaging domain resulted in a pRNA with no DNA-packaging activity but intact procapsid binding competence. Second, multiple copies of the pRNA were involved in the packaging of one genome. This higher-order dependence of pRNA in viral replication concomitantly resulted in its higher-order inhibitory effect. This finding suggested that the collective DNA-packaging activity of multiple copies of pRNA could be disrupted by the incorporation of perhaps an individual mutant pRNA into the group. Although this mutant pRNA could not be used for the inhibition of the replication of other viruses directly, the principle of using molecules with two functional domains and multiple-copy involvement as targets for antiviral agents could be applied to certain viral structural proteins, enzymes, and other factors or RNAs involved in the viral life cycle. This principle also implies a strategy for gene therapy, intracellular immunization, or construction of transgenic plants resistant to viral infection.